Pyrrolnitrin interferes with osmotic signal transduction in Neurospora crassa

Pyrrolnitrin, produced by several bacteria that are used in biological control, has an inhibitory effect on the electron transport system of respiration in Neurospora crassa. We have previously described that fludioxonil, a derivative of phenylpyrroles, affects osmotic signal transduction. Both pyrrolnitrin and fludioxonil were highly active against Botrytis cinerea, Fusarium oxysporum, Rhizoctonia solani, and N. crassa. However, a high concentration of pyrrolnitrin (more than 10 mug/ml) inhibited the growth of fludioxonil-insensitive fungi such as Pythium ultimum, Phytophthora capsici, and Saccharomyces cerevisiae. In order to clarify the difference in the antifungal mechanisms between pyrrolnitrin and fludioxonil, we observed cross-resistance in mutants of the osmotic signal transduction pathway, namely, os-1 (histidine kinase), os-4 (MAPKK kinase), os-5 (MAPK kinase), and os-2 (MAP kinase) of N. crassa. All os mutants that were resistant to fludioxonil showed cross-resistance to pyrrolnitrin without exception. The levels of resistance to pyrrolnitrin correlated well with those to fludioxonil in the 10 os-1 mutant alleles with single amino acid substitutions. However, at a concentration of 6.1 mug/ml, pyrrolnitrin inhibited the growth of all strains including the os mutants insensitive to fludioxonil even at 25 mug/ml. When the conidia of the wild-type strain were grown on a medium containing either fungicide at a concentration of 0.1 mug/ml, both fungicides induced the swelling and rupture of conidia without germ-tube formation. At a concentration of 25 mug/ml, pyrrolnitrin inhibited conidia germination without any morphological change in the fludioxonil-insensitive os-5 mutant. Both fungicides at a concentration of 1 mug/ml stimulated glycerol synthesis in the wild-type strain, but the glycerol content was reduced to a considerable extent on treatment with 25 mug/ml pyrrolnitrin. These results suggest that a primary antifungal mechanism of pyrrolnitrin against N. crassa is interference with the osmotic signal transduction pathway rather than inhibition of respiration.